Promoters control the spatial and temporal expression of genes by modulating their level of transcription. Early approaches to genetically engineered crop plants utilised strong constitutive promoters to drive the expression of foreign genes. As strategies in plant biotechnology have become more sophisticated, specific promoters have been used to target transgene expression to a particular tissue or to a particular developmental stage. The promoter of the present invention is especially versatile as it can be used either to give constitutive expression of a gene or to target increased levels of gene expression at sites of wounding or pathogen invasion.
SHH was first described, in rat liver extracts, as the activity responsible for the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to adenosine and homocysteine by the cleavage of a thioether bond in SAH [de la Haba, G. and Cantoni, G. L. (1959). J. Biol. Chem. 234, 603-608].
SAH is formed as a direct product of transmethylation reactions involving S-adenosyl-L-methionine (SAM) [Cantoni, G. L. and Scarano, E. (1954). J. Am. Chem. Soc. 76, 4744] and is known to be a potent inhibitor of most SAM mediated methyltransfer reactions. Therefore SAH is converted to homocysteine and adenosine by SHH as shown schematically below:
______________________________________ S-adenosyl-L-methionine (SAM) .uparw..dwnarw. Methyltransferase Methylated Product + S-adenosyl-L-homocysteine (SAH) .uparw..dwnarw. SHH Adenosine + L-homocysteine .uparw..dwnarw. N5-methyltetrahydrofolate Methionine ______________________________________
This pathway for the metabolism of SAH is the only pathway in most species. SHH has been found in all cells tested with the exception of Escherichia coli and other related bacteria [Shimzu, S. et al. (1984). Eur. J. Biochem. 141, 385-392].
The unique metabolic role of SHH in the removal of SAH and the structural complexity of the enzyme suggest that SHH may have a role in the regulation of the biological utilisation of SAM. SAM serves as a major methyl group donor for numerous highly specific methyltransferase reactions with a large variety of acceptor molecules; for example phenylpropanoid derivatives, cyclic fatty acids, proteins, polysaccharides and nucleic acids [Tabor, C. W. and Tabor, H. (1984). Adv. Enzymol. 56, 251-282]. It should be noted that SAM also has regulatory functions, namely the allosteric stimulation of threonine synthase. In plants, SHH has been studied primarily in relation to the biosynthesis of various phenylpropanoid derivatives.
Enzymes affecting the intracellular levels of SAH are important in the study of plant methylation reactions because it has been demonstrated that many methyltransferases are inhibited by SAH [Deguchi, T. and Barchos, J. (1971). J. Biol. Chem. 246, 3175-3181]. For example, an enzyme catalysing the methylation of caffeic acid was purified from spinach-beet leaves and found to be potently inhibited by SAH [Poulton, J. E. and Butt, V. S. (1976). Arch. of Biochem. Biophys. 172, 135-142]. Other metabolic pathways of the plant which involve transmethylation are the production of lignin and suberin, which are both derived from phenylalanine, through a series of reactions. These reactions include the methylation of caffeic acid into ferulic acid and also the methylation of s-hydroxyferulic acid into sinapic acid. Both these methylation reactions require SAM and hence produce SAH as a byproduct which needs to be removed by SHH to allow further transmethylation.
Once SHH had been isolated, many factors were calculated, such as the enzyme's pH optimum of 8.5, with a 50% activity between pH 6.5-10. Due to the Km value found for the substrate, L-homocysteine, the synthesis of SAH proceeds in vivo at a significant rate only when L-homocysteine is accumulated [Poulton, J. E. and Butt, V. S.(1976). Arch. of Biochem. Biophys. 172, 135-142].
In vivo, the adenosine produced by the hydrolysis of SAH is not deaminated but is converted to ADP by the successive action of adenosine kinase and adenylate kinase, both of which enzymes have been demonstrated in spinach-beet leaves. If L-homocysteine accumulates, it causes inhibition of SHH activity and therefore in vivo, L-homocysteine appears to be methylated by N5-methyltetrahydrofolate to methionine. Indeed, this reaction has been demonstrated in pea seedling extracts and spinach and barley leaves. Unlike all animal SHH enzymes, plant SHH is not inhibited by adenosine but is instead stabilised by low concentrations [Jakubowski, H. and Guranowski, A. (1981). Biochem. 20, 6877-6881].
The kinetic evidence shows that SHH is a sensitive regulator of SAH utilisation, its activity depending not only upon favourable concentrations of metabolites in relation to equilibrium conditions but also upon the levels of SAM, adenosine and L-homocysteine maintained within the system. These in turn will act as feed back inhibitors or activators to determine the rate of methylation reactions which are sensitive to the levels of SAH [Poulton, J. E. and Butt, V. S. (1976). Arch. of Biochem. Biophys. 172, 135-142].
As previously mentioned SHH has been found in all organisms tested except E. coli and some related species where a two step enzymatic process hydrolyses SAH into adenosine and L-homocysteine. So far the following SHH cDNAs have been isolated and published:
Rat [Ogawa, H. et al. (1987). Proc. Natl. Acad. Sci. USA. 84, 719-723], PA1 Dictostelium discoideum [Kasir, J. et al. (1988). Biochem. Biophys. Res. Commun. 153, 359-364] PA1 Human [Coulter-Karis, D. E. and Hershfield, M. S. (1989). Ann. Hum. Genet. 53, 169-175] PA1 Caenorhabditis elegans [Prasad. S. S. et al. (1991). EMBL database Accession No. M64306] PA1 Leishmania donovani [Henderson, D. M. and Ullman, B. (1992). EMBL database Accession No. M76556] PA1 Petroselinum crispum [Kawalleck, P. et al. (1992). Proc. Natl. Acad. Sci. USA. 89, 4713-4717] PA1 Rhodobacter capsulatus [Sganga, M. W. et al. (1992). Proc. Natl. Acad. Sci. USA. 89, 6328-6332]
The high level of homology between SHHs of evolutionary divergent species was highlighted further following isolation of SHH from the rat, from Dictostelium discoideum, from the purple non-sulphur photosynthetic bacterium Rhodobacter capsulatus and then from parsley (Petroselinum crispum). The bacterial SHH shows a remarkable degree of amino acid sequence homology, approximately 65% identity and 77% similarity to the previously isolated SHHs from rat, D. discoideum, human and C. elegans. This is one of the highest levels of sequence conservation ever reported between proteins having a similar function in prokaryotes and humans. Similarly, SHH cDNA from parsley is 64% identical to rat cDNA and there is 79% similarity at the amino acid level. The lack of sequence divergence between species may suggest a stringent requirement for SHH to retain its primary structure for function.
Both the R. capsulatus and the parsley amino acid sequences have an additional amino acid motif in comparison to the rat, D. discoideum, human, C. elegans and L. donovani sequences. R. capsulatus has an additional 36 amino acid region whereas parsley has an additional 41 amino acids. These two additional stretches are found in the same position in the predicted protein sequence, approximately one-third of the distance from the amino terminus. (see FIGS. 3A-3C) although they do not show significant homology.